The regulatory protein tropomyosin has been shown to confer cooperativity on the binding of myosin subfragment-1 (S01) to actin and on the actin S-1 ATPase activity. The effect of tropomyosin on the actin-activated S-1 ATPase activity and the effect of tropomyosin on the steady-state binding of S-1 to actin in the presence of ATP were measured at both low and high ratios of S-1 to actin. At low ratios of S-1 to actin, tropomyosin was found to decrease the maximum ATPase rate and to increase the actin concentration required to reach half-maximal activity by about 3-fold in each case. In addition, tropomyosin was found to have little effect on the steady-state binding of S-1 to actin in the presence of ATP. S-1 which had been extensively modified by N-ethyl malemide (NEM) exhibits a very low ATPase activity and is not dissociated from actin by ATP. We used NEM-S-1 to achieve the high ratios of bound S-1 to actin necessary to shift the tropomyosin-actin filament to the potentiated state. We then measured the ATPase of this state using a small amount of unmodified S-1. Under these conditions, tropomyosin had very little effect on the maximum ATPase rate, but the actin concentration required to reach half maximal activity was reduced about 5-fold. Little effect was observed on the steady-state binding in the presence of ATP. Thus, as tropomyosin-actin shifts from inhibited to potentiated the maximum ATPase rate is increased about 4-fold and the amount of actin required to reach half maximal activity is increased about 12-fold, while the steady-state binding of S-1 to actin in the presence of ATP remains constant. Since our previous work shows that tropomyosin may act like troponin-tropomyosin in the presence of calcium, these results suggest even after calcium activation of muscle, the number of force-producing bridges present may greatly modulate the force and velocity exhibited by a muscle fiber.